Cell growing device for in vitro cell population expansion

ABSTRACT

A cell growing device for in vitro cell population growth includes at least one hollow fiber cartridge having a plurality of capillaries at least one of which is selectively permeable. The flow of media out of a lumen of the cartridge is substantially blocked off thereby forcing media flowing into the lumen via an inflow opening to permeate across the capillaries of the cartridge and into the extracapillary space thereof.

This application is a continuation of Ser. No. 08/506,173, filed Jul.26,1995, now U.S. Pat. No. 5,627,070.

BACKGROUND OF THE INVENTION

The present invention relates to hollow fiber cartridge devices for invitro cell growth or cell population expansion.

Hollow fiber cartridges or bioreactors for in vitro cell growth are wellknown in the art and are available from several commercial sources.These devices generally have a housing and a plurality of capillaries orhollow fiber membranes. The capillaries extend between an inflow openingat one end of the cartridge and an outflow opening at the other end. Thecapillaries have selectively permeable walls through which growth mediaor culture media, carrying essential nutrients and gases, can diffuse.The interiors of the walls of the plurality of capillaries define alumen extending between the inflow and outflow openings, and the outsideof the capillaries and the housing define an extracapillary space (ECS)where cell growth or population expansion typically takes place. Thehousing generally includes one or two ports providing access to the ECSso that cells may be added or removed therefrom.

The cells are typically cultured in the ECS in growth media whichoriginates from the lumen. The media in the lumen diffuses through theselectively permeable walls of the hollow fiber membranes into the ECSto stimulate the culture or growth of the cells. To cause media to movethrough the walls of the hollow fiber membranes into the ECS, growthmedia is typically pumped through the lumen of the cartridge.

As the media passes from the inflow opening at one end of the cartridge,through the lumen, and out the outflow opening at the other end of thecartridge, a pressure drop occurs from the inflow opening to the outflowopening. At the higher pressure inlet end of the cartridge, a radialconvective flow of media moves from the lumen to the ECS bathing thecells in fresh nutrient-containing media. At the opposite end of thecartridge, near the outflow opening, media flows in the oppositedirection, thereby removing metabolic waste products and other secretedcell products from the ECS and carrying them through the hollow fibermembranes and into the lumen. These products are then carried out of thecartridge through the outflow opening. Such devices are disclosed byKnazek et al. (U.S. Pat. Nos. 3,821,087; 3,883,393; and 4,220,725) andYoshida et al. (U.S. Pat. No. 4,391,912).

In recent years, there has been an increasing demand for mammalian cellsecreted products. Mammalian cell culture is now utilized to producemany important cell products for human use including monoclonalantibodies, vaccines, lymphokines, hormones, growth factors, enzymes,and other recombinant DNA products. As these products move from researchand development, through clinical trials and to the market, a need foran economical large-scale method of production is required. The hollowfiber cell culture devices disclosed by Knazek et al. and Yoshida et al.are not suitable for large-scale manufacturing of mammalian cellsecreted products.

These devices are of limited value for large-scale cell production andmanufacturing of secreted cell products, particularly mammalian cellproducts, because of reasons related to the following: (1) therelatively large molecular weight cut-offs of the hollow fiber membraneswhich allows the required amount of oxygen to enter the ECS, but do notretain all secreted cell products or expensive serum-type nutrients; (2)inefficient oxygen diffusion to the ECS which limits cell growth; (3)formation of nutrient gradients which limit cell growth; and (4)formation of microenvironments for cell growth within the ECS whichlimits the full use of the entire capacity of the devices for cellgrowth.

The prior art devices utilize hollow fiber capillary membranes that havelarge enough molecular weight cut-offs to enable the secreted cellproducts to pass from the ECS to the lumen. This is undesirable forlarge-scale production because the secreted cell products become dilutedin the large volumes of media necessary to maintain the cells. Inaddition, serum supplements to the media are generally required on thelumen side of the device. Serum addition to large volumes of media,which are necessary for large-scale production, can be very expensiveand can also result in the extensive addition of impurities (e.g.contaminating proteins) to the already significantly diluted secretedcell products. These impurities can increase the cost of purifying thesecreted products and often result in decreased yields.

If lower molecular weight cut-off hollow fiber capillaries are utilizedin these devices, however, oxygen diffusion to the cells in the ECS willbe severely limited. The smaller the cut-off, the less oxygen diffusion.The prior art devices often include a second type of hollow fibercapillary that is especially permeable to nutrients (see e.g. Knazek etal. U.S. Pat. No. Nos. 3,821,087; 3,883,393). This is also undesirablefor large-scale production, because cells will preferentially grow onthe nutrient source capillaries, not the oxygenation capillaries,thereby decreasing the surface area in the device utilized for cellgrowth. Furthermore, the inclusion of oxygenation capillaries in thosedevices, which generally have large molecular weight cut-offs (e.g.about 0.2 microns), does not allow for the retention of secreted cellproducts or other high molecular weight proteins in the ECS.

It is desirable to retain secreted cell products in the ECS inlarge-scale production systems, because this allows the secreted cellproducts to be concentrated in the ECS, rather than being concentratedafter leaving the cartridge using one of several tedious or timeconsuming concentration procedures. It will be appreciated that highlyconcentrated products are less expensive to process and purify. It isalso desirable to retain expensive high molecular weight proteinsnecessary for cell growth (e.g. serum, growth factors, hormones and cellsecreted products) in the ECS. This would allow for the addition ofthese molecular species in relatively modest quantities to the ECS,rather than to the large lumenal media volume, which would requiresignificantly larger quantities in order to provide the necessaryconcentrations.

The prior art hollow fiber devices are also limited in their use forlarge-scale production of secreted cell products by the formation ofgradients in the ECS. When nutrient media is delivered via a motiveforce to the inflow opening of the hollow fiber device, the porousnature of the capillaries causes a change in hydrostatic pressure acrossthe length of the cartridge. Cells at the inlet or high pressure end arecontinually exposed to a convective flow of fresh nutrients and oxygen,while cells at the outlet or low pressure end are continually exposed toa concentration of metabolic waste products from cells upstream and havelimited access to fresh nutrients and oxygen. This results in theformation of a heterogeneous culture environment in the cell-occupiedECS due to the unequal nutrient distribution and the concentration ofwaste products. These nutrient gradients make it impractical toconstruct cartridges any longer than about 3-4 inches in length. Longerlengths only provide sufficient nutrients for cell growth in the highpressure inlet portion of the ECS.

Poor circulation of the media in the ECS of prior art devices can alsolead to the formation of microenvironments having widely varying cellgrowth potentials. Microenvironments can occur when pockets ofmetabolically-active cells near the inlet end of the device secretewaste products into an immediately adjacent area. The waste productsaccumulate and are not quickly removed through the outlet end of thedevice. This accumulation of waste products results in microenvironmentswhere cells are unable to grow. It will be appreciated that increasingthe length of these devices simply increases the pressure drop andresults in a worsening of the problems associated with the gradients andmicroenvironments created in the prior art devices. These problemsseverely limit the use to these devices for large-scale production ofsecreted cell products.

Knazek et al. (U.S. Pat. No. 4,184,922) disclose an improved device thatdecreases the microenvironment formation problem. This is accomplishedby weaving together two separate perfusion circuits. By altering thepressure differences between the circuits, waste products can be removedmore efficiently. However, this device still allows for the formation ofgradients, which therefore make it unsuitable for large-scaleproduction.

Cracauer et al. (U.S. Pat. No. 4,804,628) disclose an improved hollowfiber culture device that incorporates an external chamber in fluidcommunication with the ECS. By pressurizing the lumenal flow path, mediaflux to the ECS is increased. As media is forced into the ECS at theinlet end of the device, it subsequently is forced down the length ofthe cartridge to the outlet end. A percentage of the media moves intothe expansion chamber through a unidirectional valve and a percentageexits across the capillaries to the lumen as in conventional devices.When the external chamber fills, the pressure in the chamber isincreased to force the media back into the ECS through a secondunidirectional valve which directs the media to the area of the ECS nearthe inlet end of the cartridge. This cycling helps mix the ECS media,thereby minimizing gradients and microenvironments which might otherwiseexist. Oxygen diffusion is still limited, however, and gradients stilloccur.

The present invention is designed to overcome these and otherlimitations of the prior art devices and to provide a cell cultureenvironment where cells are equally perfused with nutrients, and wasteproducts are equally removed across the entire length of the hollowfiber cartridge.

SUMMARY OF THE INVENTION

In accordance with the present invention, a cell growing device for invitro cell population growth is provided wherein the cell growth occursin fluid growth media within the device. The device comprises a firsthollow fiber cartridge having a housing and a plurality of capillaries.Each of the capillaries includes walls having interiors and exteriors.The housing has a first inflow opening and a first outflow opening. Theplurality of capillaries extend between the first inflow opening and thesecond outflow opening and at least one of the capillaries hasselectively permeable walls. The interiors of the walls of the pluralityof capillaries define a first lumen extending between and being in fluidcommunication with the first inflow and the first outflow openings. Theexteriors of the walls of the plurality of capillaries and the housingdefine a first ECS. The housing has a first primary orifice in fluidcommunication with the first ECS.

The device further includes an outflow blocking mechanism forsubstantially blocking a flow of media from the first lumen via thefirst outflow opening, wherein the outflow blocking mechanism can beclosed to substantially block the flow of media from the first lumen viathe first outflow opening such that substantially all of an influx ofgrowth media into the first lumen via the first inflow opening isdirected across the capillary walls into the first ECS. Preferably, theoutflow blocking mechanism can be alternately opened and closed suchthat the flow of media from the first lumen via the first outflowopening is alternately permitted and substantially blocked. In preferredembodiments, the outflow blocking mechanism includes a first valve influid communication with the first outflow opening distal to the firstlumen, wherein the valve can be alternately opened and closed such thatthe flow of media from the first lumen via the first outflow opening isalternately permitted and substantially blocked. Preferably, the deviceincludes a controlling computer programmed to alternately switch thefirst valve open and closed.

In a preferred embodiment, a cell growing device for in vitro cellpopulation growth is provided wherein cell growth occurs in fluid growthmedia within the device. The device comprises first and second hollowfiber cartridges and a fluid connecting mechanism for fluidly connectingthe first and second cartridges. Each of the cartridges include ahousing and a plurality of capillaries. The housing of the firstcartridge including a first inflow opening and a first outflow openingand the housing of the second cartridge including a second inflowopening and a second outflow opening. The plurality of capillaries ineach cartridge extend between the respective inflow opening and outflowopenings.

At least one of the capillaries of each cartridge includes selectivelypermeable walls. The interiors of the walls of the plurality ofcapillaries in each cartridge define a lumen, the first lumen extendingbetween the first inflow and the first outflow openings and the secondlumen extending between the second inflow and the second outflowopenings. The capillaries and the housing of each cartridge define anECS, the first ECS being in the first cartridge and the second ECS beingin the second cartridge. The housing of each cartridge includes aprimary orifice in fluid communication with the ECS of each cartridge,the first primary orifice being in fluid communication with the firstECS and the second primary orifice being in fluid communication with thesecond ECS. The fluid connecting mechanism include a recirculationmechanism for recirculating fluid media from the outflow openings of therespective hollow fiber cartridges to inflow openings thereof and anextracapillary connecting mechanism for fluidly connecting the first ECSwith the second ECS, wherein all fluid communication between the firstcartridge and the second cartridge prior to passing through therecirculation mechanism passing through the extracapillary connectingmechanism.

Nutrient media is delivered to the lumen of the first cartridge via thefirst inflow opening by a motive force. The lumenal flow path is blockedafter the first outflow opening, forcing all the media to ultrafiltrateacross the capillaries to the ECS. The first primary orifice is fluidlyconnected to the second primary orifice. Therefore, the media movesthrough the first primary orifice and enters the second ECS through thesecond primary orifice. The media then moves across, or ultrafiltratesacross, the capillary membranes into the second lumenal space and flowsout the second outflow opening.

Cells can be placed in the first ECS and retained in the first cartridgeby placing a cell filter, preferably a microporous filter, after thefirst primary orifice. In this manner, all of the cells in the ECS ofthe first cartridge are continually perfused with fresh nutrients andoxygen from the ultrafiltrative flow from the lumen. Metabolic wasteproducts are removed by filtration across the capillaries in the secondcartridge. Alternately, cells can be placed in the ECS of each cartridgeand the direction of media flow reversed at preset intervals. When cellsare placed in the ECS of both cartridges, a second cell filter is placedon the second primary orifice to retain cells in the ECS of the secondcartridge.

In alternate preferred embodiments, the extracapillary connectingmechanism includes a connecting chamber in fluid communication with thefirst and second primary orifices. The connecting chamber preferablyincludes a monitoring mechanism for monitoring the presence of oxygengas (O₂) and hydrogen ion concentration (pH), a gas transfer mechanismfor exchanging gas across a membrane separating the media from acontrolled gaseous environment within the gas transfer mechanism, and agas delivery mechanism for delivering specific gases such as oxygen gas(O₂), carbon dioxide (CO₂) and nitrogen gas (N₂) to the controlledgaseous environment.

The cell growing device preferably includes an outflow valve mechanismfor alternatively preventing the flow of media from exiting via thefirst or second outflow opening respectively, an inflow valve mechanismfor alternately directing media into the lumen of the first or secondhollow fiber cartridge via the first or second inflow opening,respectively, and a control mechanism for controlling the inflow andoutflow valve mechanism such that the flow of media alternately passeseither into the first lumen via the first inflow opening or into thesecond lumen via the second inflow opening. In either event, the flow ofmedia subsequently passes through the capillary walls of the capillariesof the respective cartridge and into the respective ECS, through theconnecting mechanism, into the ECS of the other cartridge, through thecapillary walls of the capillaries the other cartridge, into the lumenand out through the outflow opening of the other cartridge. Preferably,the control mechanism include a controlling computer.

In a preferred embodiment, the cell growing device includes a pluralityof first cartridges and a plurality of second cartridges, wherein thefirst cartridges are connected in parallel with the fluid connectingmechanism and the second cartridges are connected in parallel with thefluid connecting mechanism. In other preferred embodiments, the deviceincludes a flow monitoring and restricting mechanism for individuallymonitoring and adjusting media flow to each of the hollow fibercartridges via the respective inflow openings and via the respectiveprimary orifices such that the respective flow of media therethroughinto the respective lumen or ECS of each of the cartridges can bemonitored and adjusted to maintain equal flow to each cartridge underconditions of changing resistance to media flow.

The present invention also provides methods for culturing cells in vitroin a cell growing device. One of these methods provides for culturingcells in vitro in a cell growing device wherein nutrient gradients alongthe length of hollow fiber membranes of a first hollow fiber cartridgeof the cell growing device are minimized when fluid media is pumped intothe cartridge. The method comprises the steps of culturing cells in thecell growing device wherein the device includes a media flow limitationmechanism for substantially stopping the flow of media out of the lumenvia the outflow opening of the first cartridge; and substantiallystopping the flow of media out of the first lumen via the outflowopening with the media flow limitation mechanism such that substantiallyall media flowing out of the first lumen flows into the first ECS.

An alternate method for culturing cells in vitro in a cell growingdevice including first and second hollow fiber cartridges and connectingmechanism for fluidly connecting the first and second cartridgescomprises the steps of alternately circulating media through each ofindividual first and second hollow fiber cartridges via each respectiveinflow opening in order to provide growth media for culturing cells inthe device; and alternately substantially stopping the flow of media outof the lumen of each respective hollow fiber cartridge via eachrespective outflow opening with media flow limitation mechanism whenmedia is circulated into each cartridge via its respective inflowopening such that substantially all media flowing out of the lumen outof respective cartridge flows into the ECS of the cartridge and out ofthe cartridge via a connecting mechanism connecting each first ECS witheach second ECS such that fluid can communicate therebetween.

Another alternate embodiment provides a method for culturing cells invitro in a cell growing device comprising the steps of growing andmaintaining cells in an ECS of a first hollow fiber cartridge; directingthe flow of media across the capillary walls from the lumen into the ECSof the cartridge wherein substantially all of the media flows from thelumen to the ECS and substantially all of the media flowing through thelumen and out of the cartridge via the outflow opening is blocked; andsubsequently redirecting the flow of media across the capillary wallsfrom the ECS into the lumen of the cartridge wherein the cell growingdevice includes a mechanism for monitoring and adjusting the pH of themedia in the device, and wherein the pH of the media is monitored andadjusted before the media permeates across the capillary walls into thelumen. Alternately, the cell growing device includes a mechanism formonitoring and adjusting the oxygen concentration of the media in thedevice. Preferably, the device includes a mechanism for monitoring andadjusting both the oxygen concentration and the pH of the media in thedevice.

Another method in accordance with the present invention comprises thesteps of growing and maintaining cells in a cell growing deviceincluding first and second hollow fiber cartridge, and alternatelydirecting the flow of media, first, from the lumen of the firstcartridge, across the capillary walls and into the ECS thereof, and intothe ECS of the second cartridge, and across the capillary walls into thelumen thereof, and, second, from the lumen of the second cartridge,across the capillary walls into the ECS thereof, and into the ECS of thefirst cartridge, and across the capillary walls and into the lumenthereof, wherein alternating the direction of the flow of media reversesthe flow of media from one of the cartridge to the other. Preferably,the method further comprises the step of monitoring and adjusting theoxygen concentration of the media in fluid communication with the ECS ofeither cartridge or alternately monitoring and adjusting the pH of themedia. Preferably, both the oxygen concentration and the pH of the mediaare monitored and adjusted and the method further comprises the step ofsubstantially stopping the flow of media out of the lumen of eithercartridge when the flow of media from the lumen of the respectivecartridge is directed across the capillary walls and into the ECSthereof, wherein the device includes a valve mechanism for alternatelysubstantially blocking the flow of media from the lumen of eithercartridge via each respective out flow opening.

The present invention provides many advantages over the prior art.Because the growth media pumped into any cartridge via an inflow openingcannot flow out of the lumen of the cartridge via the outflow opening,and must instead ultrafiltrate across the hollow fiber membranes toleave the lumen, the nutrient gradients along the length of the entirehollow fiber membranes are substantially eliminated or significantlyreduced. This permits consistent diffusion of nutrients to all cellsalong the entire length of the hollow fiber membranes. This, in turn,allows for consistent maximizations of cell growing potential along theentire length of the cartridge, thereby providing an advantage over theprior art.

Equal perfusion of nutrients across the entire length of the cartridge,enables larger cartridges to be utilized for the large-scale productionof secreted cell products. Perfusion of nutrients by ultrafiltration hasbeen shown to be most desirable for promoting cell growth. Smallmolecular weight cut-off fibers can also be utilized in the presentinvention to retain secreted products and other expensive growth factorsin the ECS. The monitoring mechanism for oxygen and pH, combined withthe controlled gas transfer mechanism provides a consistent homogenousenvironment for the cells and overcomes the resistance of thecapillaries to diffusion of oxygen from the lumen to the ECS. This isbecause oxygen can be added to the ECS side of the cartridge and doesnot have to diffuse to the ECS from the lumen.

The above-described features and advantages, along with various otheradvantages and features of novelty, are pointed out with particularityin the claims of the present application. However, for a betterunderstanding of the invention, its advantages, and objects obtained byits use, reference should be made to the drawings, which form a furtherpart of the present application, and to the accompanying descriptivematerial in which there is illustrated and described preferredembodiments of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a cell growing device in accordancewith the present invention having individual first and second hollowfiber cartridges.

FIG. 2 is a longitudinal cross-sectional view of the hollow fibercartridge shown in FIG. 1.

FIG. 3 is a cross-sectional view at line 3--3 of the cell growing deviceshown in FIG. 2.

FIG. 4 is a schematic diagram of a cell growing device in accordancewith the present invention having pluralities of first and second hollowfiber cartridges.

FIG. 5 is a schematic diagram of a portion of the device shown in FIG.4, wherein arrows illustrate an alternate pathway for the flow of growthmedia through the cartridges and their connectors.

FIG. 6 is a schematic diagram of a portion of the device shown in FIG.4, wherein arrows illustrate another alternate pathway for the flow ofgrowth media through the cartridges and their connectors.

FIG. 7 is a schematic diagram of a modification of the cell growingdevice shown in FIG. 4 after portions of the device have been removedand connectors and caps have been attached so that the fluid pathways inthe device may be heat sterilized.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Referring now to the drawings, and specifically to FIGS. 1, 2 and 3,FIG. 1 shows a schematic diagram of the fluid pathways of an automatedcell growth device 2 in accordance with the present invention. Thedevice 2 includes individual first and second hollow fiber cartridges 4aand 4b which are fluidly connected by a fluid connecting mechanism 5.

The hollow fiber bioreactor or cartridge 4 shown in FIG. 2 is the sameas the first and second cartridges 4a and 4b shown in FIG. 1. Eachcartridge 4 has a housing 12 and a plurality of capillaries 14. Each ofthe capillaries 14 has walls 15 having interior 15a and exteriors 15b(see FIG. 3). The housing 12 has an inflow opening 16 at one end and anoutflow opening 17 at the other end. The plurality of capillaries 14extend between the inflow opening 16 and the outflow opening 17. Each ofthe capillaries 14 preferably have selectively permeable walls 15. Theinteriors 15a of the walls 15 of the plurality of capillaries 14 in eachcartridge 4 define a lumen 20 extending between and fluidlycommunicating with the inflow and outflow openings 16 and 17. Theexteriors 15b of the capillaries 14 and the housing 12, preferablyincluding potting material 18 which binds the capillaries 14, define anECS 10 where cell growth or cell population expansion takes place. Across-sectional view of the cartridge 4 shown in FIG. 2 is shown in FIG.3.

The housing 12 includes the potting material or integral disk portions18 at each end which extend circumferentially around and thereby receivethe plurality of capillaries 14 which are bundled together. The disks 18separate the respective inflow and outflow openings 16 and 17 from theECS 10. Each cartridge 4 has two cartridge ports 24 that provide forfluid communication with the ECS 10 extends circumferentially around theplurality of capillaries 14, which are bundled together toward thecenter of the cartridge 4. Populations of cells 22 can be transferredinto the ECS 10 via either of the cartridge ports 24. It will beunderstood that such a transfer is preferably done under sterile oraseptic conditions. The ports 24 can be reversibly sealed by a septum 11to prevent contaminating material from entering the ECS 10 through theports 24. The cells 22 are injected through the septum 11 with asyringe.

Referring now also to FIG. 4, which shows a schematic diagram of apreferred cell growing device 2', which has four of each of the firstand second hollow fiber cartridges 4a' and 4b'. The plurality of firstcartridges 4a' is connected to the plurality of second cartridges 4b' ina manner which is very similar to a manner in which the first and secondcartridges 4a and 4b are connected in the smaller device 2 shown in FIG.1, except that the individual cartridges 4a' and 4b' in each pluralityof cartridges are connected to other portions of the device 2' inparallel.

The fluid connection mechanism 5 shown in FIG. 1 includes arecirculation mechanism 6 and an ECS connecting mechanism 8. The ECSconnecting mechanism 8 provides for fluid communication between the ECS10a of the first cartridge 4a and the ECS 10b of the second cartridge4b. The ECS connection mechanism 8 connects the cartridges 4a and 4b viacartridge ports 24 on each cartridge 4a and 4b, thereby providing aprimary orifice 25a and 25b for fluid communication with the ECS 10a and10b of each cartridge 4a and 4b. The primary orifice 25a of the firstcartridge 4a is connected to the primary orifice 25b of the secondcartridge 4b by ECS.

Connectors 8a made of 316 stainless connection tubing. The ECSconnectors 8a are interspersed by high molecular weight cut off filters28 which are used to retain the cells 22 in the ECS 10a and 10b of eachrespective cartridge 4a and 4b. The ECS connecting mechanism includesin-line sensors 21 and 23 for monitoring hydrogen ion concentration (pH)and oxygen gas (O₂) respectively, and a gas transfer cartridge 30 forexchanging gas across a membrane (not shown) separating growth mediafrom a controlled gaseous environment within the gas cartridge 30. Thegas transfer cartridge 30 receives gas through a sterilization filter 34from a gas delivery mechanism 32 which can deliver adjustablepercentages of CO₂, N₂, and O₂ to the controlled environment.

After the growth media flow through the filter 28 from the ECS 10, itflows through the in-line sensors 21 and 23 for pH and O₂, respectively.The in-line sensors 21 and 23 are connected to a computer controlmechanism 36, including a computer 26, and transmit information whichthe computer 26 interprets. In response, the computer control mechanism36 subsequently adjusts the relative concentrations of oxygen gas (O₂),carbon dioxide (CO₂) and nitrogen gas (N₂) which are delivered to thegas transfer cartridge 30 by the gas delivery mechanism 32.

The device 2 is automated such that the computer control mechanism 36 iscapable of alternating the direction of the flow of media through theECS connecting mechanism 8. In this way, the cells growing in the ECS 10of each of the cartridges 4a and 4b will be alternately supplied withgrowth media from the ECS connecting mechanism 8 after the pH has beenadjusted and the oxygen concentration has been replenished, therebyenhancing the growth potential of the cells.

The computer 26 is part of the computer control mechanism 36 thatincludes all of the computer connections 37 between the computer 26 andthe various elements of the device 2 which are computer controlled. Inorder to vary the direction of the flow of media through the ECSconnecting mechanism 8, the computer control mechanism 36 switches,opens or closes various valves in the recirculation mechanism 6.

The recirculation mechanism 6 includes recirculation connectors 6a thatprovide fluid pathways which interconnect the outflow openings 17a and17b of cartridges 4a and 4b to the inflow openings 16a and 16b of thesame cartridges 4a and 4b. The recirculation connectors 6a are made of316 stainless steel tubing. Also included in the recirculation mechanismare two solenoid valves 40a and 40b adjacent and proximate to theoutflow openings 17a and 17b respectively, of the respective cartridges4a and 4b. The solenoid valves 40a and 40b substantially stop the flowof media from the respective cartridge lumens 20a and 20b through therespective outflow opening 17a and 17b when they are closed. This forcesany fluid media flowing into the lumen 20 through an inflow opening 16to ultrafiltrate across the capillary walls 15 of the capillaries 14 andinto the ECS 10 when the solenoid valve 40a or 40b for one cartridge 4aor 4b or the other is closed. The solenoid valves 40a and 40b areconnected to the computer control mechanism 36. The control mechanism 36is programmed so that the solenoid valves 40a and 40b, alternatelyopened or closed such that when one is open the other will be closed.

At the same time that the solenoid valves 40a and 40b are individuallyopened and closed, the control mechanism 36 simultaneously switches athree-way valve 42 controlling the flow of media into the respectivecartridges 4a and 4b through their respective inflow openings 16a and16b such that the flow of media will be directed to one cartridge 4a or4b or the other at any particular moment. The switching of the three-wayvalve 42 and the solenoid valves 40a and 40b is coordinated such thatwhen the flow of media is directed the inflow opening 16a of the firstcartridge 4a, the solenoid valve 40a is closed to prevent the flow ofmedia from the lumen 20a from passing through the outflow opening 16a,while the solenoid valve 40b is open, thereby allowing the flow of mediafrom the lumen 20b to pass through the outflow opening 17b of the secondcartridge 4b.

Alternately, when the three-way valve directs the flow of media throughthe inflow opening 16b of the second cartridge 4b, the solenoid valve40b is closed and the solenoid valve 40a is open. In this way, thecontrol mechanism 36 directs the flow of growth media through the inflowopening 16a or 16b of one cartridge 4a or 4b and out of the outflowopening 17a or 17b of the other, thereby forcing the media to passthrough the capillary walls 15 of both cartridges 4a and 4b before themedia is recirculated back to the inflow opening 16a or 16b of eithercartridge 4a or 4b via the recirculation mechanism 6.

Because the two solenoid valves 40a and 40b are switched from open toclosed and from closed to open simultaneously, the flow of media intothe recirculation mechanism 6 can only pass into the recirculationmechanism 6 through the outflow opening 17a or 17b of one or the otherof the cartridges 4a or 4b at any one moment in time. As one solenoidvalve 40a or 40b is closing, the other is opening and the three-wayvalue 42 is switching so as to redirect the flow of recirculated growthmedia from one inflow opening 16a or 16b to the other. As this switchingtakes place the direction of the flow of media within the ECS connectingmechanism 8 is also switched.

The recirculation mechanism 6 includes the recirculation connectors 6athat connect the respective inflow and outflow openings 16 and 17 of thecartridges 4a and 4b. The recirculation connectors 6a from the outflowopenings 17a and 17b join just before reaching a centrifugal pump 44a,to form a single stainless steel pathway which carries the recirculatedmedia to the three-way valve 42. The centrifugal pump 44a pumps themedia through a regeneration mechanism 46 having similar functions tothose found in the ECS connecting mechanism 8 wherein the media isreplenished with nutrients and essential gases, and waste products fromcell growth are preferably removed. The oxygen gas (O₂) and hydrogen ionconcentration (pH) are monitored and adjusted in the regenerationmechanism 46 as previously described in relation to the ECS connectingmechanism 8. After the media is regenerated in the regenerationmechanism 46, the media then flows through a second centrifugal pump 44bto the three-way valve 42, where it is directed to the inflow opening16a or 16b of one or the other hollow fiber cartridge 4a or 4b.

The hollow fiber bioreactors or cartridges 4 are preferably commerciallyavailable hollow fiber dialysis cartridges available from CD-Medical,Inc. of Hialeah, Fla. It will be appreciated, however, that any hollowfiber bioreactors may be used whether commercially available or not.Preferably, the cartridge 4 will have a molecular weight cut-off (MWC)of about 50,000 daltons, more preferably about 30,000 daltons, even morepreferably about 15,000 daltons, and most preferably about 10,000daltons. A preferred cartridge having a 30,000 dalton MWC, is theCELL-PHARM BR 130. The most preferred cartridge, which has a 10,000dalton MWC, is the CELL-PHARM BR 110 or the CELL-PHARM Model I fromCD-Medical, Inc. of Hialeah, Fla.

The in-line sensors 21 and 23 include a pH electrode and an oxygen (O₂)electrode respectively. The information from the pH electrode and the O₂electrode is interpreted by the computer control mechanism 36 andutilized to adjust the mixture of O₂, N₂ and CO₂ delivered to the gastransfer cartridge 30 by the gas delivery mechanism 32. Similarfunctions are effected by similar elements in the regeneration mechanism46. The media directed toward the cartridge 30 and away from thecartridge 30 is monitored by these in-line sensors 21 and 23 in thefluid pathway. The ability to reverse the flow of media passing throughthe ECS connecting mechanism 8, and the ability to adjust the pH andoxygen levels therein, among other things, enhance nutrient perfusionand oxygen delivery so that a cell population of about 1×10⁹, preferablybetween approximately 5×10⁹ and 10×10⁹ cells can be supported in eachcartridge 4, and between approximately 5×10⁹ and 10×10⁹, preferablybetween approximately 4×10¹⁰ and 10×10¹⁰ cells can be supported by thepreferred device shown in FIG. 4.

Referring now also to FIG. 4, the automated cell growth device 2' shownin FIG. 4 has similar fluid dynamics to that of the less complicateddevice 2 shown in FIG. 1. The plurality of first hollow fiber cartridges4a' are connected in parallel with one another as are the plurality ofsecond hollow fiber cartridges 4b'. Like the simpler service 2 shown inFIG. 1, the outflow openings 16' are connected to a recirculationmechanism 6'. The flow of media from the lumens 20' through the outflowopenings 16' of either group of parallel cartridges 4a' and 4b' arecontrolled by solenoid valves 40a' and 40b' respectively, which are, inturn, connected to and controlled by the computer control mechanism 36'.The fluid dynamics of the industrial scale cell growing device 2' arecontrolled by the control mechanism 36' in a manner similar to orparallel to the way they are controlled in the smaller device 2 shown inFIG. 1. The computer 26' is connected to solenoid valves 40a' and 40b'and the three-way valve 42' which each correspond to similar elements ofthe simpler device 2.

The biggest difference in the industrial scale device 2', as compared tothe smaller device 2, is the fact that a plurality of cartridges 4' areconnected to the system in parallel in place of the single cartridges 4aand 4b. The flow of media from the lumens 20' through the outflowopenings 16' can be alternately stopped by the control mechanism 36' byalternately closing one or the other solenoid valve 40a' and 40b'.Similarly, the three-way valve 42', which is also controlled by thecomputer control mechanism 36', can be switched to alternate the flow ofmedia directed to the inflow openings 16a' and 16b' of one group ofparallel cartridges 4a' or 4b' or the other.

The fluid connecting mechanism 5' of the industrial scale cell growingdevice 2' includes the recirculation mechanism 6' and the ECS connectingmechanism 8' that each have functions which are equivalent to thecorresponding mechanisms 6 and 8 in the smaller device 2. The cells 22'are retained in the ECS 10' by a filter 28' that allows the media topass out of the primary orifice 25' at a relatively high flow rate. Thefilter 28' can be any device which allows fluids to pass, but retainscells (e.g. a 5.0 micron filter, large molecular weight cut-offmicroporous cartridge, or the like). Because there are a plurality offirst cartridges 4a' and a plurality of second cartridges 4b', however,each group of which are connected in parallel with the other members ofthat group, a flow monitoring and restricting mechanism 51 forindividually monitoring and adjusting media flow to each of thecartridges 4' via either the respective inflow openings 16' or therespective primary orifices 25' are provided. The flow monitoring andrestricting mechanisms 51 include a flow meter or sensor 51a to measurethe media flow rate through the fluid path and a needle valve 51b whichare connected to the control mechanism 36' are preferably adjusted inresponse to the information registered on the flow meter 51a.Alternately, the needle valve 51b may be adjusted manually.

Because the needle valve 51b only allows flow in one direction and isonly used to monitor and adjust the flow into the cartridges, the ECSconnecting mechanism 8' of the industrial scale device 2' is somewhatmore complex than the corresponding mechanism 8 of the smaller device 2.Media flow leaving the ECS 10' of the first cartridges 4a' does not passthrough the flow monitoring and restricting mechanisms 51. Instead, whenmedia leaves the ECS 10' through the ECS connectors 8a', it is shuntedto a bypass pathway 52 in the ECS connecting mechanism 8' when the mediareaches the flow monitoring and restricting mechanisms 51. The bypasspathway 52 is connected with an ECS three-way valve 54 that is switchedsimultaneously with the recirculation three-way valve 42', and thesolenoid valves 40a and 40b by the computer control mechanism 36'.

The industrial scale device 2' includes a growth media reservoir 63 thatis preferably a 10 liter tank constructed from 316 stainless steel. Thetank 63 is wrapped with resistance coils 64 that can generate heat toraise the media temperature above the ambient temperature which ispreferably below 37° C. during cell culture operations. Temperaturemonitoring mechanisms 73 are attached to the stainless steel connectingtubes or connectors 8a' and 6a' at various points, and also to thereservoir 63, to ensure that a proper temperature is reached during anin-line sterilization procedure discussed hereinbelow.

The hydrogen ion concentration (pH) and oxygen (O₂) concentration of thegrowth media in the reservoir 63 is monitored by a pH electrode 65 andan oxygen electrode 66 that are inserted into the reservoir throughports 61 in the reservoir 63. The oxygen concentration (O₂) and hydrogenion concentration (pH) in the growth media 60 in the reservoir 63 isconstantly adjusted by circulating the media 60 through a high speedsecondary circulation loop 67. The media 60 leaves the reservoir 63under the motive force of a centrifugal pump 44c' is primed by closingan in-line valve 59 and using a syringe to inject media into the loop 67through an in-line port 57 located between the valve 59 and pump 44c' inthe secondary loop 67. The loop 67 includes a gas transfer cartridge 74which is preferably identical to the gas cartridge 30' in the ECSconnecting mechanism 8'.

The gas delivery mechanism 32' is connected to the computer controlmechanism 36', as are the probes 65 and 66 and the pump 44c'. Inresponse to the information regarding pH and oxygen concentrationreceived by the computer 26' from the respective probes 65 and 66, thespeed of the pump 44c' and the mixture of gases delivered to the gascartridge 74 by the gas delivery mechanism 32' are adjusted.Alternately, pump 44c' can be adjusted manually so that the media ispumped at a rate of up to about 400 ml/min or greater. In that event,information regarding the flow rate through the gas transfer cartridge74 is taken into account during programming of the computer controlmechanism 36'. The computer control mechanism 36' controls the mixtureof gas leaving the gas delivery mechanism 32' by simultaneouslycontrolling the flow of gas to the gas cartridge 74 from three gassupply reservoirs 81, 82 and 83, which supply oxygen gas (O₂), carbondioxide (CO₂) and nitrogen gas (N₂), respectively.

The gas flow from each reservoir 81, 82 and 83 is monitored and adjustedby a flow monitoring and adjusting mechanism 80 which includes a flowmeter or sensor 80a, a variable adjustment valve 80b, and a steppermotor 80c. All of these elements are connected to the computer controlmechanism 36' which integrates the information received therefrom, andadjusts the stepper motors 80c to adjust the valves 80b so that a propermixture of gas is delivered to the gas cartridge 74. The gas from thereservoirs 81, 82 and 83 is mixed into a common passageway after beingpassed through a 0.2 micron sterilization filter 70, and then bubbledthrough aqueous media in a reservoir 68a to humidify the gas. The gas isthen passed through a second sterilization filter 70 and into the gascartridge 74. The gas can leave the cartridge 74 through a secondsterilization filter 70. Each of the gas cartridges 30, 30' and 74 haveheating elements 75 that allow the temperature in each gas cartridge 30,30' or 75 to be raised above the ambient temperature. It will beappreciated that any gas transfer cartridge may be used, whethercommercially available or not, so long as it meets the needs of thepresent invention. A preferred gas transfer cartridge, however, is theCELL-PHARM Hollow Fiber Oxygenator, which can be obtained fromCD-Medical, Inc., Hialeah, Fla.

After the pH and the oxygen concentration levels of the media 60 areadjusted in the gas transfer cartridge 74, the media is returned to thereservoir 63. In addition to providing an efficient system to control pHand oxygen concentration (O₂), the secondary circulation loop 67 alsoserves to keep the media 60 circulating within the reservoir 63. The gasdelivery mechanism 32' in the secondary circulation loop 67 issubstantially the same as the gas delivery system 32' in the ECSconnecting mechanism 8'. Preferably, the regeneration mechanism 46 inthe less complicated cell growing device 2 shown in FIG. 1, also has agas delivery system that is similar or identical to the system providedby the industrial scale cell growing device 2'.

Fresh media is slowly added to the growth media 60 in the growth mediareservoir 63 from a fresh media reservoir 68b. The fresh media is drawnout of the reservoir 68b through a first flexible tube 84 that isconnected to a first rigid tube 77 which extends into the mediareservoir 63. The flexible tube 84 passes through a first peristalticpump 79a that pumps the fresh media from the fresh media reservoir 68binto the growth media reservoir 63 at a variable rate. As the freshmedia leaves the reservoir 68b, air is drawn into the reservoir 68bthrough a 0.2 micron sterilization filter 70. At the same time, a secondflexible tube 85 that is connected to a second rigid tube 87 whichextends into the media reservoir 63, withdraws growth media 60 when thelevel of the media 60 in the reservoir 63 reaches a height equal to orgreater than the lower end 87a of the second rigid tube 87. The media 60is withdrawn under the motive force of a second peristaltic pump 79b andis delivered into a spent media reservoir 68c. The reservoir 68c isequipped with a sterilization filter 70 that allows gas to escape as thereservoir 68c is filled with media removed from the growth mediareservoir 63. In this way, the media is constantly replenished withfresh media, thereby removing waste products and replenishing thenutrients needed for continued cell growth.

Media is withdrawn from the media reservoir 63 to supply the cartridges4' through a stainless steel tube 86 under the motive force of acentrifugal pump 44b'. This pump 44b' is primed in the same manner asthe centrifugal pump 44c' in the secondary circulation loop 67. Athree-way valve 42' then directs the growth media alternately to a bandof first hollow fiber cartridges 4a' or a bank of second hollow fibercartridges 4b' via their respective inflow openings 16a' and 16b'.

Referring now also to FIGS. 5 and 6, the growth media passing throughthe three-way valve 42' will follow two alternate pathways. When thethree-way valve 42' directs the growth media to the inflow openings 16a'of the plurality of first hollow fiber cartridges 4a', the fluid pathwill follow the path delineated by the arrows shown in FIG. 5. In such acase, the solenoid valve 40a' is closed, preventing the flow of mediafrom the lumen 20a' of the plurality of first cartridges 4a'. When thesolenoid valve 40a' is closed, the solenoid valve 40b' is open so thatmedia can flow from the lumens 20b' of the plurality of secondcartridges 4b' through their respective outflow openings 17b' and intothe recirculation mechanism 6'. In such a case, the flow of media in theECS connecting mechanism 8' is directed from the ECS 10a' of the firstcartridges 4a' to ECS 10b' of the second cartridges 4b'.

When the flow of media through the three-way valve 42' is directedthrough the inflow opening 16b' of the plurality of second hollow fibercartridges 4b', the direction of the media flow in the ECS connectingmechanism 8' is reversed as is shown in FIG. 6. In that situation, thesolenoid valve 40b' is closed and the solenoid valve 40a' is open sothat the media may enter the recirculation mechanism 6' after it passesout of the lumens 20a' of the first cartridges 4a' through the outflowopenings 17a' thereof.

Media entering either the plurality of first or second hollow fibercartridges 4a' or 4b', which each contain four cartridges 4' in thepreferred embodiment, is split into four fluid flow paths which flowthrough a flow monitoring and restricting mechanism 51 prior to enteringeach respective cartridge 4'. Each flow monitoring and restrictingmechanism 51 includes a flow meter or sensor 51a and a needle valve 51b.The flow rate to each cartridge 4a' or 4b' may be adjusted by adjustingthe needle valve 51b for each fluid path. This provides a mechanism foradjusting the flow to each of the individual hollow fiber cartridges 4'.This is necessary because each cartridge 4' has a different resistanceto flow. Media will take the path of least resistance. If no adjustmentswere possible, one cartridge could receive more flow than another andcells in the other cartridge 4' could be denied the favorable growthenvironment afford the cells in the cartridge 4' get the greatest flow.Furthermore, as cells grow asynchronously in the various individualcartridges 4', changes in resistance occur over time and requireadjustment of the flow to assure equal perfusion in each cartridge 4' sothat balanced cell growth may occur. It will be appreciated, that eachplurality of cartridges 4', may include any practical number ofcartridges permitted by the fluid dynamics of any alternate embodimentsthe cell growing device 2' of the present invention.

The solenoid valves 40a' and 40b' force an influx media coming into alumen 20' from a respective inflow opening 16' to ultrafiltrate throughthe hollow fiber membranes or capillary walls 15' into the ECS 10' whenthey are closed. The ultrafiltrative flow rate is controlled byincreasing or decreasing speed of the centrifugal pump 44b. Preferably,constant lumenal pressure is maintained. The lumenal pressure ismonitored by in-line fluid pressure sensors 72. Preferably, all thevariable aspects of the industrial scale device 2' are automated orcontrolled by the computer control mechanism 36' which may be suppliedwith an emergency power back-up system (not shown). The control system36' preferably switches the three-way valves 42' and 54', and switchesthe solenoid valves 40a' and 40b' from open to closed and from closed toopen, simultaneously on a regular schedule. Preferably the cycleswitches, and the direction of the flow in the ECS connecting mechanism8' reverses every ten minutes. Alternately, aspects of the device, whichcould otherwise be controlled by the computer, may be manually adjusted.

Independent control of pH and oxygen concentration (O₂) is accomplishedby controlling the concentration of (CO₂) and (O₂) entering theindividual gas cartridges 30, 30' and 74. A computer controlled steppermotor 80c is mechanically connected to each of the needle valves 80bcontrolling the flow rates of each of the three gases. The (CO₂)concentration is raised to lower pH and lowered to raise pH. The N₂concentration is adjusted up or down to maintain a constant flow rate ofthe three gas mixture. However, if the (CO₂) concentration reaches zeroand the pH is still falling, the computer 26' will increase the gas flowrate by proportionately increasing N₂ and O₂ flow rates. This will allowa greater amount of (CO₂) to diffuse out of the media, thereby loweringthe pH thereof. If this is not sufficient to maintain the pH at adesired level, the rate at which fresh media is fed into the mediareservoir 63 is increased either manually or, preferably, as directed bythe computer control mechanism 36'. Oxygen (O₂) concentration iscontrolled in a similar manner. As more O₂ is demanded, the computer 26'will increase the O₂ flow rate and decrease the N₂ flow rate.

The cells 22 that are to be expanded in the cartridges 4 are inoculatedthrough the septum 11 on the port 24 in the housing 12 with a syringe.If cells are the final product, they are harvested from the cartridges 4by utilizing a Haemonetics V50 aphoresis instrument. A custom tubing setis connected to the inoculation port 24 of each cartridge or bioreactor4. Cells 22 are forced out of the cartridges 4 under ultrafiltrativeforce. The cells are captured in the centrifuge of the aphoresisinstrument, while the flushing media is diverted to a 10 liter wastebag. The cells 22 may then be washed and resuspended for further use.This cell harvest procedure has the advantage of being a completelyclosed system, thereby minimizing the risk of contamination.

Referring now also to FIG. 6, the device 2' may be prepared for anin-line sterilization procedure by removing certain parts that cannotwithstand high temperatures and replacing them with temporary parts thatcan. For instance, the hollow fiber cartridges 4' and adjacent filters28', and the gas transfer cartridges 30, 30' and 74 are removed andreplaced by stainless steel tubing parts 92 and 93, respectively, whichare designed to fit the spaces left unconnected by the removal of theaforementioned parts. Once the stainless steel tubing parts 92 and 93are in place, and the flexible tubes 84 and 85 have been removed and thecaps 84b and 85b are in place, steam is pumped into the inlet valve 90at 15 psi. The steam is pumped in through a sterilization steam filter78 and is allowed to circulate through the various fluid pathways byclosing in-line valve 59a and opening the outlet valve 58 which isequipped with a sterilization steam filter 78. The steam is preferablygenerated in a commercial steam generator which preferably generatessteam from low-endotoxin water for injection (e.g. H200 generator fromFinn Agua, Inc., Seattle, Wash.). The sterilization steam filters 78through which the steam enters and leaves the system are preferably 0.2micron steam filters.

A pressure gauge 91 makes it possible to monitor the steam pressure sothat a pressure of 15 psi may be maintained. In addition, a pressurerelief valve 62, having a 15 psi pressure relief rating and equippedwith a sterilization steam filter 78, is provided to insure that thepressure does not exceed 15 psi. The steam is allowed to circulatethroughout the system. Temperature is preferably monitored atapproximately 18 inch intervals, or at each contortion in the variouspathways, by the temperature monitoring mechanisms 73. Steam ispreferably circulated at 15 psi for 30 minutes. Temperature is monitoredand the time is not counted unless all temperature monitors exceed 121°C. In an alternate embodiment the sterilization process is automated andcomputer controlled.

Preferably, the hollow fiber cartridges 4 of the present invention havea length greater than about four inches, more preferably greater thanabout five inches and even more preferably greater than about sixinches. Alternate embodiments of the present invention may be equippedwith hollow fiber cartridges 4 which have a length of about eight inchesor more. It will be appreciated that, when used with the presentinvention, hollow fiber cartridges 4 having even greater lengths willprovide even greater space for cell growth. Therefore, it is consideredto be desirable to have hollow fiber cartridges 4 which may even exceeda length of about 10-12 inches.

It will be appreciated that any type of cell which can grow in a cellculturing device, can be cultured or grown in the cell growing devicesof the present invention. The cells which may be grown in the cellgrowing devices of the present invention include, but are not limited tothe following classes of cells: mammalian cells, plant cells,microbiological cells and other single cell organisms, such as bacteria,fungi, and algae, and the like. The mammalian cells include anchoragedependent cells or suspension or floating cells. These cells may beprimary cells, transformed cells, neoplastic cells, cells altered byrecombinant DNA techniques, fused cells, including mammalian cells fusedwith other types of cells or with other mammalian cells, cells whichhave been otherwise altered by natural or artificial means, and thelike. The plant cells include normal plant cells, transformed orotherwise altered cells, cells altered by recombinant DNA techniques,fused cells and the like. The microbial or other single cell organismsalso include a variety of transformed or otherwise altered cellsincluding, but not limited to, cells or organisms altered by recombinantDNA techniques, fusion, or the like.

Although the present invention has been described with reference topreferred embodiments, workers skilled in the art will recognize thatchanges may be made in form and detail without departing from the spiritand scope of the invention.

What is claimed is:
 1. A cell growing device for in vitro cellpopulation growth, the cell growth occurring in fluid growth mediawithin the device, the device comprising:a first hollow fiber cartridgehaving a housing and a plurality of capillaries, each of the capillariesincluding walls having interiors and exteriors, the housing having afirst inflow opening and a first outflow opening, the plurality ofcapillaries extending between the first inflow opening and first outflowopening, at least one of the capillaries having selectively permeablewalls, the interiors of the walls of the plurality of capillariesdefining a first lumen extending between and being in fluidcommunication with the first inflow and the first outflow openings, theexteriors of the walls of the plurality of capillaries and the housingdefining a first extracapillary space, the housing having a firstprimary orifice in fluid communication with the first extracapillaryspace; an outflow blocking mechanism for substantially blocking a flowof media from the first lumen via the first outflow opening, wherein theoutflow blocking mechanism can be closed to substantially block the flowof media from the first lumen via the first outflow opening such thatsubstantially all of the influx of media into the first lumen via thefirst inflow opening is directed across the capillary walls into thefirst extracapillary space; and an extracapillary space monitoringmechanism for monitoring and directly adjusting the oxygen concentrationand the pH of the media located in the first extracapillary space. 2.The cell growing device of claim 1, wherein the outflow blockingmechanism can be alternately opened and closed such that the flow ofmedia from the first lumen via the first outflow opening is alternatelypermitted and substantially blocked.
 3. The cell growing device of claim2, wherein the outflow blocking mechanism includes a first valve influid communication with the first outflow opening distal to the firstlumen, wherein the first valve can be alternately opened and closed suchthat the flow of media from the first lumen via the first outflowopening is alternately permitted and substantially blocked.
 4. The cellgrowing device of claim 3, wherein the device includes a controllingcomputer, the controlling computer being programmed to alternatelyswitch the first valve open and closed.
 5. The cell growing device ofclaim 4, wherein the extracapillary space monitoring mechanism includesa gas transfer mechanism for exchanging gas across a membrane separatingthe media from a controlled gaseous environment within the gas transfermechanism, the device including a gas delivery mechanism for deliveringspecific gases to the controlled gaseous environment, the specific gasesincluding oxygen gas, carbon dioxide, nitrogen gas, or combinationsthereof.
 6. The cell growing device of claim 1, wherein at least one ofthe capillaries having the selectively permeable walls has a molecularweight cut-off of equal to or less than about 30,000 daltons.
 7. Aprocess for the in vitro growth of cells comprising:depositing a firstplurality of cells in a first extracapillary space of a first hollowfiber cartridge, wherein the first hollow fiber cartridge has a housingand a plurality of capillaries, each of the capillaries including wallshaving interiors and exteriors, the housing having a first inflowopening and a first outflow opening, the plurality of capillariesextending between the first inflow opening and the first outflowopening, at least one of the capillaries having selectively permeablewalls, the interiors defining a first lumen extending between and beingin fluid communication with the first inflow opening and the firstoutflow opening, the exteriors and the housing defining the firstextracapillary space, the housing having a first primary orifice influid communication with the first extracapillary space; supplying thefirst plurality of cells with nutrient media through a first flow pathextending from the first inflow opening to the first primary orifice;and oxygenating the first plurality of cells by flowing oxygenated mediathrough a second flow path extending from the first primary orifice tothe first outflow opening, wherein substantially blocking the flow ofmedia from the first lumen via the first outflow opening and causessubstantially all of the influx of media into the first lumen via thefirst inflow opening to be directed across the capillary walls into thefirst extracapillary space.
 8. The process of claim 7, wherein a firstvalve in fluid communication with the first outflow opening distal tothe first lumen alternately permits and substantially blocks flow ofmedia from the first lumen via the first outflow opening.
 9. The processof claim 8, and further comprising:depositing a second plurality ofcells in a second extracapillary space of a second hollow fibercartridge, wherein the second hollow fiber cartridge has a housing and aplurality of capillaries, each of the capillaries including walls havinginteriors and exteriors, the housing having a second inflow opening anda second outflow opening, the plurality of capillaries extending betweenthe second inflow opening and the second outflow opening, at least oneof the capillaries having selectively permeable walls, the interiorsdefining a second lumen extending between and being in fluidcommunication with the second inflow opening and the second outflowopening, the exteriors and the housing defining the secondextracapillary space, the housing having a second primary orifice influid communication with the second extracapillary space; supplying thesecond plurality of cells with nutrient media through the second flowpath further extending from the second inflow opening to the secondprimary orifice; and oxygenating the second plurality of cells byflowing oxygenated media through the first flow path further extendingfrom the second primary orifice to the second outflow opening.
 10. Theprocess of claim 9, wherein a second valve in fluid communication withthe second outflow opening distal to the second lumen alternatelypermits or substantially blocks flow of media from the second lumen viathe second outflow opening.
 11. The process of claim 10, and furthercomprising fluidly connecting the first and second hollow fibercartridges with a fluid connecting mechanism, the fluid connectingmechanism including a recirculation mechanism for recirculating fluidmedia from the respective outflow openings of the hollow fibercartridges to inflow openings thereof and an extracapillary spaceconnecting mechanism for fluidly connecting the first extracapillaryspace with the second extracapillary space, wherein the recirculationmechanism includes the first valve and the second valve, and wherein allfluid communication between the first cartridge and the second cartridgeother than that passing through the recirculation mechanism passesthrough the extracapillary space connecting mechanism.
 12. The processof claim 11, wherein the extracapillary space connecting mechanismincludes a connecting chamber in fluid communication with the firstprimary orifice and the second primary orifice, wherein the connectingchamber provides fluid communication between the first extracapillaryspace and the second extracapillary space, and wherein the connectingchamber including a monitoring mechanism for monitoring the oxygenconcentration and pH of the media therein.
 13. The process of claim 12,and further comprising exchanging gas across a membrane separating themedia from a controlled gaseous environment for delivering specificgases to the cells, wherein the specific gases includes oxygen gas,carbon dioxide, nitrogen gas, or combinations thereof.
 14. The processof claim 10, and further comprising controlling the operation of thefirst and second valves using a controlling computer, wherein thecontrolling computer is programmed to simultaneous open one of the firstand second valves and close the other, thereby alternately switching thefirst and second valves from open to closed and from closed to open in areciprocal relationship wherein one of the first and second valves isalways open and one is always closed.
 15. The process of claim 14,wherein a third valve alternately directing media from the recirculationmechanism to the first lumen or second lumen via the respectiveproximate inflow opening, the third valve being connected to andcontrolled by the controlling computer, the controlling computer beingprogrammed such that the media from the recirculation means isalternately directed to either the first lumen or second lumen at thesame time that the flow of media from that respective lumen issubstantially blocked by the closure of the respective first valve orthe second valve.
 16. The process of claim 7, and further comprisingmonitoring and adjusting the oxygen concentration and the pH of themedia located in the first extracapillary space.
 17. The process ofclaim 7, wherein the selectively permeable walls have a molecular weightcut-off of equal to or less than about 30,000 daltons.
 18. A process forthe in vitro growth of cells comprising:depositing a first plurality ofcells in a first extracapillary space of a first hollow fiber cartridge,wherein the first hollow fiber cartridge has a housing and a pluralityof capillaries, each of the capillaries including walls having interiorsand exteriors, the housing having a first inflow opening and a firstoutflow opening, the plurality of capillaries extending between thefirst inflow opening and the first outflow opening, at least one of thecapillaries having selectively permeable walls, the interiors defining afirst lumen extending between and being in fluid communication with thefirst inflow opening and the first outflow opening, the exteriors andthe housing defining the first extracapillary space, the housing havinga first primary orifice in fluid communication with the firstextracapillary space; depositing a second plurality of cells in a secondextracapillary space of a second hollow fiber cartridge, wherein thesecond hollow fiber cartridge has a housing and a plurality ofcapillaries, each of the capillaries including walls having interiorsand exteriors, the housing having a second inflow opening and a secondoutflow opening, the plurality of capillaries extending between thesecond inflow opening and the second outflow opening, at least one ofthe capillaries having selectively permeable walls, the interiorsdefining a second lumen extending between and being in fluidcommunication with the second inflow opening and the second outflowopening, the exteriors and the housing defining the secondextracapillary space, the housing having a second primary orifice influid communication with the second extracapillary space; supplying thefirst plurality of cells with nutrient media through a first flow pathextending from the first inflow opening to the first primary orifice;oxygenating the second plurality of cells by flowing oxygenated mediathrough the first flow path extending from the second primary orifice tothe second outflow opening; supplying the second plurality of cells withnutrient media through a second flow path extending from the secondinflow opening to the second primary orifice; and oxygenating the firstplurality of cells by flowing oxygenated media through the second flowpath extending from the first primary orifice to the first outflowopening.
 19. The process of claim 18, and further comprising fluidlyconnecting the first and second hollow fiber cartridges with a fluidconnecting mechanism, the fluid connecting mechanism including arecirculation mechanism for recirculating fluid media from therespective outflow openings of the hollow fiber cartridges to inflowopenings thereof and an extracapillary space connecting mechanism forfluidly connecting the first extracapillary space with the secondextracapillary space, wherein all fluid communication between the firstcartridge and the second cartridge other than that passing through therecirculation mechanism passes through the extracapillary spaceconnecting mechanism.
 20. The process of claim 19, wherein a first valvein fluid communication with the first outflow opening distal to thefirst lumen alternately permits or substantially blocks flow of mediafrom the first lumen via the first outflow opening and wherein a secondvalve in fluid communication with the second outflow opening distal tothe second lumen alternately permits or substantially blocks flow ofmedia from the second lumen via the second outflow opening.
 21. Theprocess of claim 20, and further comprising controlling the operation ofthe first and second valves using a controlling computer, wherein thecontrolling computer is programmed to simultaneous open one of the firstand second valves and close the other, thereby alternately switching thefirst and second valves from open to closed and from closed to open in areciprocal relationship wherein one of the first and second valves isalways open and one is always closed.
 22. The process of claim 21,wherein a third valve alternately directing media from the recirculationmechanism to the first lumen or second lumen via the respectiveproximate inflow opening, the third valve being connected to andcontrolled by the controlling computer, the controlling computer beingprogrammed such that the media from the recirculation means isalternately directed to either the first lumen or second lumen at thesame time that the flow of media from that respective lumen issubstantially blocked by the closure of the respective first valve orthe second valve.
 23. The process of claim 19, wherein theextracapillary space connecting mechanism includes a connecting chamberin fluid communication with the first primary orifice and the secondprimary orifice, wherein the connecting chamber provides fluidcommunication between the first extracapillary space and the secondextracapillary space, and wherein the connecting chamber including amonitoring mechanism for monitoring the oxygen concentration and pH ofthe media therein.
 24. The process of claim 23, and further comprisingexchanging gas across a membrane separating the media from a controlledgaseous environment for delivering specific gases to the cells, whereinthe specific gases includes oxygen gas, carbon dioxide, nitrogen gas, orcombinations thereof.